Preterm delivery is the chief problem in obstetrics today, affecting 10% of all births1. It accounts for more than 70% of perinatal mortality and nearly half of long-term neurological morbidity, especially in infants who are born at less than 32 weeks of gestation and weigh less than 1,000 g. With ˜60% of these newborns developing respiratory distress syndrome (RDS) and a 50% lethality, RDS is the leading cause of neonatal mortality2. RDS results from insufficient production of surfactant by immature type 2 pneumocytes in preterm infants, but can also result from dysfunction or deficiency of surfactant in term infants due to inherited mutations, meconium aspiration, hemorrhage, infections and others3. Surfactant is a mixture of phospholipids and surfactant-associated proteins (SP-A to SP-D), which lowers surface tension at the air-water interface and thereby prevents alveolar collapse and respiratory failure. Surfactant phospholipids are synthetized from substrates, provided by glycogen stores in fetal immature pneumocytes4. Neonatal intensive care has improved the survival of infants with RDS, but often at the expense of the development of bronchopulmonary dysplasia or chronic lung disease of prematurity5. Treatment with oxygen may irreversibly damage lung parenchyma and angiogenesis, while prenatal steroid treatment causes neurological, metabolic, cardiovascular and hormonal side-effects, and impairs growth6. Surfactant treatment is effective, but expensive and only symptomatic7.
Interactions between branching airways and blood vessels are critical for normal lung development8. A major factor in lung vascular development is vascular endothelial growth factor (VEGF), which binds its receptors Flk-1 (VEGF-R2) and Flt-1 (VEGF-R1)9. Three VEGF-isoforms exist: a diffusable VEGF120, a matrix-bound VEGF188 and VEGF164, which can bind matrix and is still diffusable. VEGF is deposited at the leading edge of branching airways, where it stimulates vascularization10. Indirect evidence suggests, however, that VEGF also affects epithelial growth and differentiation. Type 2 pneumocytes and bronchiolar epithelial cell produce VEGF and possess VEGF receptors11,12. VEGF levels are also considerably higher in the bronchoalveolar fluid than in the blood12, suggesting that epithelial cells affect their own function by releasing VEGF into the airway lumen. Remarkably, the lung is one of the few organs where VEGF levels remain elevated in the adult, even though no active angiogenesis occurs. Previous studies provided circumstantial evidence for a role of VEGF in lung development, but did not provide functional in vivo proof for a role of VEGF in lung maturation and surfactant production. For instance, VEGF levels in tracheal aspirate were lower in infants with lung immaturity developing bronchopulmonary dysplasia than in those surviving without pulmonary complications in some13-15 but not in other studies16. Exogenous VEGF stimulates growth of epithelial cells in early embryonic lung explants in vitro17, but the relevance of endogenous VEGF for lung maturation just prior to birth in vivo and the possible therapeutic potential of VEGF in preventing RDS in preterm infants remain unknown. In the present invention we show that loss of HIF-2α causes fatal RDS in newborn due to insufficient surfactant production. We show that VEGF plays an important role in lung maturation since VEGF levels are reduced in HIF-2α deficient mice, neonates expressing only the VEGF120 isoform or with impaired HIF-2α-dependent VEGF expression die of RDS, and intra-amniotic administration of anti-Flk-1 antibodies aggravated lung prematurity. Importantly, intra-uterine delivery of VEGF before birth or intra-tracheal injection of VEGF after birth stimulates conversion of glycogen to surfactant, improved lung function and prevented RDS in premature. In summary, one aspect of the invention shows the use of VEGF for the manufacture of a medicament to treat RDS in premature infants.
A second aspect of the invention deals with the manufacture of a medicament to treat and/or to prevent pulmonary hypertension. Hypoxia causes proliferation of pulmonary vascular cells, in contrast with the usual growth-suppressive effect of hypoxia on most other cell types. Chronic hypoxic conditions are known to induce pulmonary vascular remodeling and subsequent pulmonary hypertension and right ventricular hypertrophy, thereby constituting a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD). Although several molecules such as endothelin-1 and platelet derived growth factor (PDGF) are believed to play an important role during pulmonary hypertension, the precise molecular mechanisms of this process are still elusive. It has been shown that the transcription factor HIF-1α is involved in the physiological response to chronic hypoxia. Heterozygous HIF-1α± mice showed delayed polycythemia and right ventricular hypertrophy and impaired pulmonary hypertension and vascular remodeling after exposure to chronic hypoxia, indicating a significant role for HIF-1α in the development of pulmonary hypertension. HIF-1α was originally cloned as a basic helix-loop-helix transcription factor, mediating the cellular adaptation to hypoxia. During hypoxia HIF-1α upregulates the expression of a number of genes involved in erythropoiesis, glycolysis and angiogenesis by formation of a heterodimer with HIF-1β (also termed aryl hydrocarbon receptor nuclear translocator; ARNT), which binds to a hypoxia-response element (HRE) in the promoter of these target genes. In addition, HIF-1α has also been implicated in the induction of apoptosis in hypoxic and hypoglycaemic conditions. Recently, a novel hypoxia-inducible factor, HIF-2α (also known as EPAS-1, HLF, HRF or MOP2) was identified, which is also able to bind to hypoxia-response elements after heterodimerization with HIF-1β. Although HIF-2α is a homologue of HIF-1α, the role of HIF-2α in glycolytic, angiogenic, apoptotic or possible disease processes is unknown and unpredictable. In the present invention, we have examined the endogenous role of HIF-2α by targeted gene-inactivation in murine embryonic stem (ES) cells. It has been found that HIF-2α is a new therapeutic target for the treatment of pulmonary hypertension.
(A) Top, targeting vector pPNT.HIF-2α; middle, map of the wild type (WT) gene; bottom, homologously recombined (HR) HIF-2α allele. Analytical restriction digests and hybridization probes A (0.6-kb NcoI-EcoRV fragment) and B (2.3-kb NheI-EcoRI fragment) for genotyping are indicated. (B) Southern blot analysis (probe A) of StuI-digested genomic DNA from ES cells generating a 7-kb WT and 7.5-kb HR HIF-2α allele. (C) RTPCR analysis of total RNA of ES cells for HIF-2α gene expression. HPRT gene expression was used as an internal control. (D) Immunoblot analysis on total cell extract from WT, HIF-1α−/− and HIF-2α−/− ES cells for HIF-2α gene expression during normoxia (N) and hypoxia (H).